The PI's laboratory has recently identified five trans-acting regulatory factors governing prokaryotic gene expression in response to changes in oxygen tension and light intensity. During the proposed funding period, each of these regulatory factors will be genetically and biochemically characterized for their role in controlling gene expression in response to oxygen and light. Two "oxygen responding" regulatory proteins contain sequence motifs indicating that they are membrane spanning sensor kinases that transmits information about anaerobioses via phosphorylation of a DNA binding response regulator. Each of these sensor kinases have sequence motifs indicating that they may be involved in forming an oxygen labile iron sulfur complex. The role of iron binding in kinase function will be probed by performing site directed mutagenesis on critical iron binding ligands as well as by EPR analysis of isolated polypeptides. One of these sensor kinases (SenC) is also structurally similar to the ScoI protein in yeast mitochondria which is known to be involved in assembly of cytochrome c oxidase. As is observed in yeast, mutations in SenC leads to a reduction in cytochrome c oxidase synthesis. It should be noted that in humans the failure to properly synthesize a functional cytochrome c oxidase complex has been implicated in degenerative diseases such as Parkinson's and Ischemic heart disease. An additional regulatory protein (HvrA) has been identified which is responsible for controlling gene expression in response to changes in light intensity. The light sensing capabilities of this protein will be analyzed by assaying for flavin binding capabilities of HvrA. The laboratory is also characterizing a light sensing regulatory protein involved in controlling expression of the enzyme S-adenosyl homocysteine hydrolase (SAHase). In humans, SAHase is an important contributing factor in the cytotoxic effects of adenosine leading to severe immune deficiency in patents with adenosine deaminase (ADA) deficiency.